A cloned segment of the African green monkey (Cercopithecus aethiops) genome that contains DNA sequences homologous to the control region of simian virus 40 is being studied. This sequence, 450 base pairs in length, is embedded in a genomic DNA region that is especially rich in interspersed repeated sequences. The segment homologous to SV40 is flanked by two members of the Alu family. The SV40-like region, which is hypersensitive to DNase I in monkey chromatin, serves as a transcriptional start site in both possible directions for cellular RNA synthesis. Also, the sequence provides information for initiation of transcription from vectors constructed by molecular cloning as measured by expression of an E. coli gene after transfection of the vector into mammalian cells. Expression was measured both by the percent of cells transformed by the E. coli gene and by analysis of messenger RNA transcribed from the vector. Multiple transcriptional start sites were detected in both directions by S1 nuclease analysis. Some of these coincide with the start sites mapped for the genomic transcripts. The SV40-like region also is a bidirectional transcriptional start site in in vitro reactions using fractionated cell free extracts. In vitro transcription depends on the presence of a fraction that is also required for in vitro transcription from SV40 DNA itself but not for other host cell promoters tested. The data suggest that there is a special class of cellular promoters that like SV40 promoters depend on the presence of a short G-rich DNA segment (5'=GGGCGGPuPu) and interact with a specific factor, Sp 1.